1. Introduction
Nuclear morphology changes characteristic of apoptosis appear within the cell together with a distinctive biochemical event: the endonuclease-mediated cleavage of nuclear DNA. In fact, formation of DNA fragments of oligonucleosomal size (180-200 bp) is an hallmark of apoptosis in many cell types.
The present protocol provides a method for DNA separation of fragmented and intact DNA fractions and for their analysis by agarose gel electrophoresis. In apoptotic cells specific DNA cleavage becomes evident in electrophoresis analysis as a typical ladder pattern due to multiple DNA fragments. However, although this protocol is simple and generally able to provide good results, it is only qualitative because of its limitations in DNA recovery and solubilization. In order to obtain a cleaner DNA, other methods for DNA preparation are required (in some cases use of proteinase K for deproteinization is recommended).
2. Protocol
Cell suspension at 1-5x106 cells/ml in complete RPMI medium (A1)
TTE solution: TE buffer pH 7.4 (A1) with 0.2% Triton X-100 (store at 4°C)
NaCl 5M, ice cold
Isopropanol, ice cold
Ethanol at 70%, ice cold
TE buffer pH 7.4 (A1)
Loading buffer 10x (A1)
TBE buffer for electrophoresis (A1)
Ethidium bromide solution (A1)
Electrophoresis-grade agarose
DNA molecular weight markers
Refrigerated cell centrifuge (A3)
Microfuge (A3)
Heating block (A3)
Gel electrophoresis apparatus (A3)
DC power supply (A3)
UV transilluminator (A3)
Polaroid Camera + films (A3)
2.2. Methodology
1. Dispense 0.5 ml of cell suspension (no less than 5x105, otherwise DNA will not be detectable by photography of ethidium bromide stained gel, and no more than 5x106, to avoid difficult handling of too high amounts of insoluble DNA) in tubes labeled B (bottom).
2. Centrifuge cells at 200xg at 4°C for 10 min.
3. Transfer supernatants carefully in new tubes labeled S (supernatant).
4. Add to the pellet in tubes B 0.5 ml of TTE solution and vortex vigorously. This procedure allows the release of fragmented chromatin from nuclei, after cell lysis (due to the presence of Triton X-100 in the TTE solution) and disruption of the nuclear structure (following Mg++ chelation by EDTA in the TTE solution).
5. To separate fragmented DNA from intact chromatin, centrifuge tubes B at 20,000xg for 10 min at 4°C.
6. Carefully transfer supernatants in new tubes labeled T (top).
7. Add to the small pellet in tubes B 0.5 ml of TTE solution.
8. Add to the 0.5 ml volume present in tubes B, S and T, 0.1 ml of ice-cold 5M NaCl and vortex vigorously. The addition of the salt should be able to remove histons from DNA.
9. Add to each tube 0.7 ml of ice-cold isopropanol and vortex vigorously.
10. Allow precipitation to proceed overnight at -20°C. This step can be shortened by putting samples in a bath of ethanol/dry ice for 1 hr.
11. After precipitation, recover DNA by pelleting for 10 min at 20,000xg at 4°C.
12. Discard supernatants by aspiration or by rapidly inverting tubes and carefully remove any drops or fluid remaining adherent to the wall of the tube with a paper towel corner. This can be a critical step because the pellet could be loosen and transparent, hard to be seen.
13. Rinse the pellets by adding to each tube 0.5-0.7 ml of ice-cold 70% ethanol.
14. Centrifuge tubes at 20,000xg for 10 min at 4°C.
15. Discard supernatants by aspiration or by rapidly inverting tubes. Carefully remove any drops or fluid remaining adherent to the wall of the tube by inverting tubes over an absorbent paper towel for 30 min. Let air dry the tubes in upright position for at least 3 hr before proceeding.
16. Dissolve DNA by adding to each tube 20-50 m l of TE solution and place the tubes at 37°C for 1-3 days. The redissolution of DNA may be a crical step, in fact it depends on the DNA quantity and size present in the samples. Thus, the non-fragmented DNA contained in the B tubes, may need higher volumes of TE and longer incubation times in order to be resuspended.
17. Mix the samples of DNA with loading buffer by adding 10x loading buffer to a final concentration of 1x. The addition of loading buffer to samples allows to load gel wells more easily and to monitor the run of samples.
18. Place samples in a heating block at 65°C for 10 min and immediately load 10-20 m l of them to each well of a standard 1% agarose gel containing ethidium bromide 0.5 mg/ml. Appropriate DNA molecular weight markers should be included. Ethidium bromide is a potential carcinogen: wear gloves and handle with care.
19. Run the electrophoresis in standard TBE buffer after setting the voltage to the desired level. During electrophoresis it is possible to monitor the migration of samples by following the migration of bromophenol blue dye contained in the loading buffer.
20. Stop the electrophoresis when the dye reaches about 3 cm from the end of the gel.
21. To visualize DNA, place the gel on a UV transilluminator and take photos of the gel. Wear eye and skin protection when UV are on.
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